Caloric Restriction (CR) is the only well-established protocol in mammals that consistently increases lifespan and delays age-related physiological declines. The recent finding that the health benefits of CR apply to rhesus monkeys has increased interest in developing interventions that can mimic the health benefits of CR. However, the molecular basis of the CR effects in aging is poorly understood. We and others have postulated that a profound metabolic reprogramming underlies the health benefits of CR. Recent studies suggest that the mitochondrial sirtuin Sirt3 plays a major role in mitochondrial metabolic control, and we have shown that the ability of CR to prevent age-related hearing loss is completely dependent on Sirt3. Based on these and other observations, our central hypothesis is that Sirt3 mediated deacetylation of key metabolic targets in response to CR increases resistance to oxidative stress and as a consequence prevents age-related mitochondrial dysfunction. Within this application we propose to extensively and mechanistically investigate one of the major processes underlying the effects of CR at the organismal level, the mitochondrial adaptations in response to reduced caloric intake. The proposed studies will address the effects of Sirt3-mediated mitochondrial adaptations in response to CR at the biochemical level through the analysis of Sirt3 targets in mitochondria, at the cellular level through the analysis of age-related mitochondrial dysfunction, and at the tissue-specific level through cardiac and skeletal muscle functional assays of aged animals. We will also determine if the ability of CR to increase lifespan and prevent age-related pathology requires Sirt3. Results from the study will provide a detailed molecular understanding of mitochondrial adaptation to CR and the role of sirtuins in these pathways; Knowledge of the molecular mechanisms and key pathways regulated has potential for significantly improving health outcomes through the development of novel therapeutics that specifically target these pathways.